In the past few years, advances in imaging methods have made it possible to track cell movement within 3 dimensional tissues, opening up a new frontier in understanding the in vivo control of cell migration. The thymus of the mouse is an ideal experimental system to explore this problem, since developing T cells are highly motile and undergo long range migrations that are tightly linked to their developmental program. Our general approach consists of three phases. 1) We will use 2-photon imaging to observe and quantitate thymocyte migration deep within the thymus and to form initial hypotheses about how thymocyte motility is regulated. 2) We will refine our hypotheses using stochastic computer simulations to assess how factors such as attractive/repulsive molecules, adhesion proteins, inherent motility and cell packing could effect thymocyte migration. 3) We will then test our hypotheses by experimentally altering these factors and examining the effect of these alterations on thymocyte migration and TCR repertoire selection. This fundamental information about the process whereby immature thymic precursors give rise to mature, functional T cells, will improve our ability to manipulate the immune response to disease and to improve human health.